Recent evidence has linked the use of 3,4-methylenedioxymethamphetamine (MDMA) to persistent serotonergic neurotoxicity in humans. Direct injection of MDMA into the brain does not result in neurotoxic effects observed after peripheral administration, suggesting systemic metabolism of MDMA plays an important role in the development of neurotoxicity. Since synthetic glutathione conjugates of MDMA have been shown to cause neurotoxicity after being taken up into the brain via the GSH transporter, this project hypothesizes that catecholamine metabolites of MDMA produced by P450 enzymes are oxidized to orthoquinones, which react readily with nucleophilic glutathione to form thioether conjugates in the liver. It is these thioether adducts of MDMA that are believed to cause serotonergic toxicity following systemic MDMA administration. The objectives of this project are to show direct evidence of the formation of glutathione adducts of MDMA and to identify the enzymes and mechanism involved in the formation of these neurotoxic glutathione metabolites in humans. Specific aims of this proposed project are to: (1) identify and quantitate the thioether metabolites of MDMA formed during in vitro incubation of fresh human hepatocytes with MDMA; (2) identify the specific class and subfamily of glutathione S-transferase (GST) enzymes involved in the conjugation of the catecholamine o-quinone metabolites of MDMA to glutathione; (3) characterize the enzymatic and nonenzymatic mechanism involved in these glutathione conjugation reactions in humans. Solid phase extraction, LC-MS and LC-MS/MS analytical techniques will be utilized to separate, identify and quantitate the thioether metabolites of MDMA formed during in vitro incubation of MDMA with fresh hepatocytes from human donors. To identify the enzyme and to elucidate the catalytic mechanism involved in the formation of glutathione conjugates of MDMA, various biochemical approaches will be applied, including immunoinhibition, substrate inhibition, and immunoblotting studies. A kinetic enzyme study with GSTs will also be performed to identify the specific GST isozymes and to elucidate the mechanism involved in the conjugation of the o-quinone metabolites of MDMA with GSH. The goal of this project is to generate valuable information about the thioether metabolites formed from MDMA in humans, and to ultimately help ascertain the underlying mechanism through which MDMA produces neurotoxicity.